Razvoj mlečnokislinske bakterije Lactococcus lactis s sposobnostjo dostave proteinskih ligandov receptorja za interlevkin 23

Benko, Anja (author), Berlec, Aleš (mentor)

Kronično vnetno črevesno bolezen (KVČB) delimo na ulcerozni kolitis ter Crohnovo bolezen. Za obe obliki je vzrok prekomerna aktivacija imunskega odziva v sluznici črevesja, pri čemer pomembno vlogo igra citokinska os IL-23/Th17. Trenutno zdravljenje poteka sistemsko z aminosalicilati, imunosupresivi, kortikosteroidi, pri najhujših oblikah pa s protitelesi proti različnim tarčam v procesu vnetnega odziva. Ker imajo protitelesa visoko ceno, veliko neželenih učinkov ter pri velikem deležu bolnikov niso učinkovita, poteka razvoj majhnih proteinov z visoko afiniteto vezave, specifičnostjo ter sposobnostjo inhibicije vnetja. V okviru magistrskega dela smo uporabili rekombinantne proteine REX009, REX115 in REX125 s potrjenim antagonističnim delovanjem na receptor za IL-23 in jih nadgradili s tarčno dostavo. Kot dostavni sistem smo izbrali mlečnokislinske bakterije Lactococcus lactis, saj imajo status GRAS (Generally recognized as safe) in pripomorejo k vzpostavitvi normalne mikrobiote v sluznici črevesja. Cilj je bil pripraviti bakterije s sposobnostjo izločanja proteinov REX v gojišče in bakterije s sposobnostjo površinske predstavitve proteinov REX. Za pripravo plazmidov z genskimi konstrukti REX smo uporabili plazmide pNZ8148, pSDBA3b in pGEM-T Easy. Končni plazmidi so bili pripravljeni v ogrodju plazmida pSDBA3b, ki omogoča z nizinom nadzorovano izražanje genov, z dodanimi geni za signalni peptid Usp45, označevalni peptid Flag, protein REX ter površinsko sidro cAcmA. V procesu priprave končnih konstruktov smo genske fragmente pomnoževali s PCR, jih ločevali z agarozno gelsko elektroforezo ter jih v plazmide vstavljali z restrikcijo in ligacijo. Plazmide smo analizirali s postopkom PCR na kolonijah in z določanjem nukleotidnega zaporedja ter s preverjenimi transformirali bakterije. Izražanje proteinov REX smo preverili po ločevanju s postopkom NaDS PAGE, in sicer s metodama prenosa po westernu ter z barvanjem z barvilom Commassie Brilliant Blue. Izločanje proteinov REX ter obseg njihove površinske predstavitve smo preverili s pretočno citometrijo. Potrdili smo uspešno vezavo na receptor za IL-23 za vse površinsko predstavljene proteine REX, pri čemer smo opazili najmočnejšo vezavo s proteinom REX125. Ob tem smo ugotovili tudi negativen vpliv sklopitve z označevalnim peptidom Flag na obseg vezave. Dodatno smo opravili modificiran test ELISA, s katerim smo potrdili prisotnost izločenih proteinov REX v gojišču in njihovo sposobnost vezave na imobiliziran receptor IL-23R, pri čemer je največji obseg vezave izkazal protein REX009. V raziskavi smo tako potrdili zmožnost rekombinantnih bakterij za dostavo vezalcev receptorja IL-23R, njihovo terapevtsko učinkovitost pa bo potrebno ovrednotiti na živalskem modelu KVČB.

• Language: Slovenian
• Keywords: Lactococcus lactis, kronična vnetna črevesna bolezen, površinska predstavitev, rekombinantni proteini
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-113277
• Publication date in RUL: 18.12.2019

Secondary language

• Language: English
• Title: Development of lactic acid bacterium Lactococcus lactis with the ability to deliver interleukin 23 receptor binding proteins

Abstract

Inflammatory bowel disease is a term for two conditions, ulcerative colitis and Crohn's disease, that are characterized by excessive activation of the immune response in the intestinal mucosa, in which the IL-23/Th17 axis plays an important role. The disease is treated systemically with aminosalicylates, immunosuppressants, corticosteroids and, in the most severe forms, with antibodies against various targets in the process of inflammatory responses. Because antibodies have high prices, many side effects, and they are not effective in a large proportion of patients, development of smaller proteins with high binding affinity, specifity and ability of inhibition was neccesary. In the context of the master's thesis we used recombinant proteins REX009, REX115 and REX125 with a confirmed antagonist activity for the receptor for IL-23 and upgraded them with a targeted delivery. For the delivery system we selected the lactic acid bacterium Lactococcus lactis, because of its GRAS status (generally recognized as safe) and beneficial effects on gut microbiota. We aimed to engineer recombinant L. lactis cells secreting REX proteins into the media, and with the ability to display REX proteins on surface. For the preparation of plasmids with the REX gene construct we used model plasmids pNZ8148, pSDBA3b and pGEM-T Easy. The final plasmids were constructed within the plasmid framework of pSDBA3b, which allows nisin controlled gene expression, with the addeded Usp45 secretion signal, Flag tag, REX protein, and the cAcmA surface anchor. In the process of preparing constructs, gene fragments were amplified with PCR, separated by agarose gel electrophoresis, and inserted into the plasmids by restriction and ligation. Plasmids were analyzed with colony PCR, verified with nucleotide sequencing and introduced into L. lactis. Expression of recombinant proteins was demonstrated with SDS PAGE, Western blot and Commassie Brilliant Blue staining . To evaluate the secretion and the surface display, we used flow cytometry. We confirmed the successful binding of the IL-23 receptor to surface displayed REXs, with the highest binding ability demonstrated for REX125. Furthermore, we investigated the effect of fusion with the Flag peptide, and found a negative effect on the extent of binding. In addition, statistically significant binding of secreted REX009 and REX115 proteins to bacterially produced soluble human IL-23R was confirmed by ELISA.

• Keywords: Lactococcus lactis, inflammatory bowel disease, surface display, recombinant proteins